Serum vitamin B12 assay and kit therefor

ABSTRACT

In a vitamin B 12  assay, serum, containing vitamin B 12  bound to endogenous binders therefor, is released from such endogenous binders by a releasing agent comprised of a water miscible organic liquid, such as, acetone, reducing agent and cyanide ions. Such release can be effected at room temperature, as compared to prior procedures which required heating to elevated temperatures.

This invention relates to the release of serum vitamin B₁₂ fromendogenous binders therefor, and more particularly, to an improved assayand kit for vitamin B₁₂.

Currently, vitamin B₁₂ is measured by a competitive protein bindingtechnique. In brief, competitive protein binding for the assay ofvitamin B₁₂ involves the ability of unlabeled vitamin B₁₂ in serum orother media to compete with labeled vitamin B₁₂ for a specific vitaminB₁₂ binder, and thereby inhibit the binding of labeled vitamin B₁₂. As aresult of the competitive inhibition, the ratio of bound labeled vitaminB₁₂ to free labeled vitamin B₁₂ diminishes as the concentration ofunlabeled vitamin B₁₂ is increased. Accordingly, the concentration ofvitamin B₁₂ in an unknown sample; e.g., a patient's serum, is obtainedby comparing the inhibition observed with that produced by known amountsof vitamin B₁₂, as presented in a standard curve. In determining vitaminB₁₂ in a serum, it is necessary to initially release the vitamin B₁₂from endogenous binders therefor. Currently, such a release is effectedby heating the serum to elevated temperatures for a period of time;e.g., temperature in the order of about 100° C. for about 15 minutes orlonger. Such heating increases the length of the assay, necessitates theuse of a water bath, and in addition requires cooling before thecompetitive protein binding technique can be initiated.

In accordance with one aspect of the present invention, serum vitaminB₁₂ is released from endogenous binders therefor by use of a liquidreleasing agent which includes an organic liquid, which does not destroyvitamin B₁₂, a reducing agent which does not destroy vitamin B₁₂, andcyanide ions.

In accordance with another aspect of the present invention, there isprovided an improved assay for vitamin B₁₂ in a serum wherein thevitamin B₁₂ is released from endogenous binders by the use of a liquidreleasing agent which includes an organic liquid which does not destroyvitamin B₁₂, a reducing agent which does not destroy vitamin B₁₂ andcyanide ions.

In accordance with a further aspect of the present invention there isprovided a kit for the assay of vitamin B₁₂ which includes vitamin B₁₂tracer, vitamin B₁₂ binder and a liquid releasing agent for releasingvitamin B₁₂ from endogenous binders which is comprised of an organicliquid which does not destroy vitamin B₁₂, a reducing agent which doesnot destroy vitamin B₁₂, and cyanide ions.

Applicant has found that by the use of a liquid releasing agent inaccordance with the invention it is possible to release vitamin B₁₂ fromendogenous serum proteins without using the elevated temperaturesheretofore required to effect heat release of the vitamin B₁₂. Thus,vitamin B₁₂ can be released from endogenous binder at temperatures lowerthan 100° C.; e.g., it is possible to effect heat release at roomtemperature, although higher temperatures could be employed.

The organic liquids which are used in the B₁₂ releasing agent of thepresent invention are preferably water miscible to facilitate thesubsequent assay. In addition, the organic liquid should be one whichwhen diluted in the subsequent assay does not materially affect the B₁₂binding capacity of the B₁₂ binder employed in the assay; i.e., thebinder is capable of providing sufficient B₁₂ binding capacity in thepresence of the assay diluted organic liquid to provide an effective B₁₂assay. The organic liquid is generally one of which is known in the artto be suitable for use as a water miscible organic solvent. Asrepresentative examples of suitable organic liquids, there may bementioned: ketones, such as acetone; alcohols; in particular alkanolssuch as methanol, ethanol, etc., ethers, such as dioxane,tetrahydrofuran, etc.; and miscellaneous liquids such asdimethylformamide, dimethylsulfoxide, etc. It is to be understood thatthe releasing agent may include one or more of such organic liquids. Theselection of a particular liquid is deemed to be within the scope ofthose skilled in the art from the teachings herein.

The reducing agent employed as one of the components of the vitamin B₁₂releasing agent may be either an organic or inorganic reducing agentwhich does not destroy vitamin B₁₂. As representative examples ofsuitable reducing agents there may be mentioned: dithiothreitol,dithioerythritol, monothioglycol, thiodiglycol, thioglycollic acid,cysteine, homocysteine, glutathione, mercaptoethanol, sulfhydrylreducing agents or inorganic reducing agents such as sodium sulfite,sodium dithionate, sodium sulfide, sodium metabisulfite, with theorganic reducing agents being preferred. The reducing agent componentcould be a reducing agent which is normally known to be suitable for thedestruction of vitamin B₁₂ in that, in some cases, in the amountsemployed in the releasing agent of the present invention, such reducingagents do not destroy vitamin B₁₂. As a result, such reducing agents areincluded within the scope of the present invention.

The cyanide ion component is provided by a suitable cyanide compoundsuch as a cyanide salt; e.g., potassium cyanide.

The three components are employed in an amount effective to denature B₁₂endogenous serum proteins and to release vitamin B₁₂ therefrom. Ingeneral, the ratio of reducing agent to organic liquid is from 0.00001:1to 0.1:1, preferably from 0.00025:1 to 0.00075:1. The ratio of cyanideion to organic liquid is generally from 0.00001:1 to 0.1:1, andpreferably from 0.00025:1 to 0.00075:1 The selection of optimum amountsis deemed to be within the scope of those skilled in the art from theteachings herein.

The releasing agent may be employed as an aqueous solution of theorganic liquid, reducing agent and cyanide ions. In such cases, theratio of organic liquid to water is generally from 4:1 to 6:1.

The releasing agent is added to the serum in an amount effective todenature vitamin B₁₂ -binding endogenous protein and to release vitaminB₁₂ therefrom. In general, the releasing agent is added to provide aratio of organic liquid to serum of at least 0.5:1, and preferably atleast 1.28:1, with the ratio of organic liquid to serum generally beingno greater than 10:1. The use of excess amounts of releasing agent mayadversely affect the subsequent B₁₂ assay, and as a result, thereleasing agent is preferably not employed in quantities which are muchgreater than the effective minimum amount for release of vitamin B₁₂from the endogenous binder.

The serum is contacted with the releasing agent for a time sufficient torelease vitamin B₁₂ from endogenous binder. In general, the contactingtime is about 15 minutes, although longer times could be employed. Thevitamin B₁₂ may be released from endogenous binder at room temperature,although higher temperatures could be employed; e.g., up to 37° C.

The vitamin B₁₂ releasing agent may be incorporated into a kit for theassay of vitamin B₁₂ which includes, in addition to the releasing agent,a vitamin B₁₂ binder and a vitamin B₁₂ tracer. The kit may furtherinclude suitable standards, buffer(s) and an adsorbent for separatingunbound B₁₂ and B₁₂ tracer, from B₁₂ and B₁₂ tracer bound to B₁₂ binder.

The vitamin B₁₂ binder may be either a naturally occurring binder or anantibody to vitamin B₁₂, produced by a procedure known in the art forraising antibodies. As representative examples of natural binders, theremay be mentioned: saliva, chicken serum, intrinsic factor, etc., withintrinsic factor being preferred. The selection of a suitable binder isdeemed to be within the scope of those skilled in the art from theteachings herein.

The binder may be unsupported or supported on a suitable support for usein a so-called solid phase assay. Thus, for example, the binder may beincluded in the kit adsorbed on or covalently bound to a solid support;e.g., a tube, solid particles, sheet, etc.

The vitamin B₁₂ tracer is vitamin B₁₂ or appropriate analog thereof (onewhich is specifically bound by the B₁₂ binder) labeled with a suitable"tag" or "label." The label or tag, as known in the art, may be aradioisotope, an enzyme or a fluorescent material. In general, thetracer is radiolabeled; in particular, ⁵⁷ Co. The tracer could also be aradioiodinated analog of vitamin B₁₂ ; e.g., as disclosed in U.S. Pat.No. 3,981,863.

In accordance with the vitamin B₁₂ assay, the serum is incubated withthe releasing agent, preferably at room temperature for a timesufficient to denature the serum B₁₂ endogenous binders and releasevitamin B₁₂ therefrom. Subsequently, the serum sample is diluted with asuitable buffer (the pH of the assay may vary over a wide range of pH),with such dilution minimizing the affect of the releasing agentcomponents on the binder employed in the assay. The sample is contactedwith vitamin B₁₂ tracer and binder, and the unbound tracer and B₁₂ isseparated from the B₁₂ and tracer bound to the B₁₂ binder. In the casewhere the B₁₂ binder is not supported on a solid support, the free B₁₂and free tracer may be separated by the use of a particulate adsorbent;e.g., dextran coated charcoal, ion exchange resin, etc.

The following example is illustrative of the invention; however, thescope of the invention is not to be limited thereby:

EXAMPLE

The following example of the assay is one in which the assay isperformed at pH 9.3. It may also be done at any pH known in the art.

The following reagent kit is used in the assay.

1. Tracer

0.75 u Ci Vitamin B₁₂ [⁵⁷ Co], human serum albumin, sodium borate,dextran and preservatives.

2. Binder

Hog Intrinsic Factor formulated for a trace binding of 55±15%, humanserum albumin, dextran and preservatives.

3. Standards

Containing human serum albumin, sodium borate, sodium chloride andpreservatives.

3A Standard A--Zero level

3B Standard B--100 pg/ml B₁₂

3C Standard C--200 pg/ml B₁₂

3D Standard D--400 pg/ml B₁₂

3E Standard E--1000 pg/ml B₁₂

3F Standard F--2000 pg/ml B₁₂

4. Releasing Agent

82.5% Acetone: 17.5% water solution which contains 0.05% PotassiumCyanide and 0.05% dithiothreitol.

5. Buffer

pH 9.3 Sodium Borate with 6.25 μg Potassium Cyanide/ml.

6. Dithiothreitol Solution 5%

6A. Assay Buffer

A mixture of 1 ml of Reagent 6 to 50 ml of Reagent 5.

7. Dextran Coated Charcoal Suspension

4.4±0.1 G dextran charcoal dry mix (1:10), suspending agent and sodiumchloride in 100 ml distilled water.

    __________________________________________________________________________     PROTOCOL                                                                     __________________________________________________________________________    Preparation of a Standard Curve                                                                  Clinical Determinations                                    __________________________________________________________________________      Number 16 polypropylene tubes                                                                  1.                                                                              Starting with 17, consecutively                            sequentially from 1-16.                                                                          number two polypropylene tubes                                                for each clinical sample.                                  Add Standards (Reagents                                                                        2.                                                                              Add 100 μl patient sample to                            3A-3F) as follows: each of two tubes. Mix gently.                           __________________________________________________________________________                            Vitamin B.sub.12                                      Tube No.      Standard  as pg/ml                                              __________________________________________________________________________    3-6           100 μl A                                                                             0                                                     7, 8          100 μl B                                                                             100                                                    9, 10        100 μl C                                                                             200                                                   11, 12        100 μl D                                                                             400                                                   13, 14        100 μl E                                                                             1000                                                  15, 16        100 μl F                                                                             2000                                                  __________________________________________________________________________      Add 150 μl of releasing                                                                     3.                                                                              Add 150 μl releasing reagent                            reagent (reagent 4) to tubes                                                                     (reagent 4) to all tubes.                                  3-16.                                                                         Incubate all tubes at room temperature for 15 minutes.                        Add Assay Buffer (Reagent 6A)                                                                  5.                                                                              Add 1000 μl Assay Buffer                                as follows:        (Reagent 6A) to each tube.                               __________________________________________________________________________            Tube No.    Buffer Volume                                             __________________________________________________________________________            1, 2        1600 μl                                                        3-16        1000 μl                                                __________________________________________________________________________      Add 100 μl Tracer (Reagent 1)                                                               6.                                                                              Add 100 μl Tracer (Reagent 1)                           to all tubes. Mix gently by                                                                      to each tube. Mix gently by hand.                          hand. Set tubes 1 and 2 aside                                                 at room temperature until Step 14.                                            Add 100 μl Binder (Reagent 2)                                                               7.                                                                              Add 100 μl Binder (Reagent 2)                           to tubes 5- 16. Gently vortex.                                                                   to each tube. Gently vortex.                             From this point, all tubes are treated as follows:                              Incubate at room temperature for 45 minutes from the time of the              last addition of the binder. Cover the rack of tubes with aluminum            foil to exclude light or keep in the dark.                                    Add 0.4 ml dextran-coated charcoal to tubes 3-16 and to all                   patient sample tubes (17, 18, etc.). Do not add to tubes 1 and 2.             This reagent is "squirted"  into each tube to obtain a uniform                suspension                                                                    in the reaction mixture.                                                    10.                                                                             Keep at room temperature for 10 minutes from the time of last                 addition in Step 9.                                                           Centrifuge at a minimum of 1240 × g for 15 minutes, preferably          in                                                                            the cold. Shorter times may be sufficient in equipment of higher              centrifugal force.                                                            Consecutively number a set of clean tubes, beginning with 3.                  Gently decant each clear supernatant into the similarly numbered              tube prepared in Step 12. Maximal transfer is obtained by hitting             the rims together. Avoid decanting over any charcoal to the                   counting tube. Discard the charcoal residues.                                 Count the radioactivity in the supernatants and tubes 1 and 2 in              sequence for one or more minutes with a scintillation (gamma)                 counter.                                                                    __________________________________________________________________________

The Standard Curve covers the range of 100 to 2000 pg/ml of Vitamin B₁₂.A "Blank" (tubes 3 and 4) is used to correct for background counts andradioactive tracer which is not adsorbed onto the charcoal.

This invention is particularly advantageous in that it permits releaseof vitamin B₁₂ from endogenous binders without the necessity of heatingto elevated heat releasing temperatures. The ability to effect suchrelease at lower temperatures; e.g., room temperature, shortens theoverall assay procedure, and it eliminates the previous heating andcooling step required during such assays.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

We claim:
 1. A process for the release of vitamin B₁₂ from endogenousserum binders, comprising:contacting serum containing vitamin B₁₂ boundto endogenous binders therefor with a vitamin B₁₂ releasing agentcomprising a water miscible organic liquid solvent which does notdestroy vitamin B₁₂, a reducing agent which does not destroy vitaminB₁₂, and cyanide ions, said releasing agent being employed in an amounteffective to release vitamin B₁₂ from endogenous binders therefor tothereby eliminate the necessity for heating the serum to heat releasingtemperatures.
 2. The process of claim 1 wherein the ratio of reducingagent to organic liquid solvent is from 0.00001:1 to 0.1:1 and the ratioof cyanide ion to organic liquid solvent is from 0.00001:1 to 0.1:1. 3.The process of claim 2 wherein the organic liquid solvent is selectedfrom the group consisting of ketones, alcohols, ethers,dimethylformamide and dimethylsulfoxide.
 4. The process of claim 3wherein the cyanide ion to organic liquid ratio is from 0.00025:1 to0.00075:1 and the reducing agent to organic liquid ratio is from0.00025:1 to 0.00075:1.
 5. The process of claim 4 wherein the releasingagent is employed as an aqueous solution wherein the ratio of organicliquid to water is from 4:1 to 6:1.
 6. The process of claim 4 whereinthe releasing agent is employed in an amount to provide a ratio oforganic liquid to serum of at least 0.5:1 and no greater than 10:1. 7.The process of claim 6 wherein the organic liquid is acetone and thereducing agent is dithiothreitol.
 8. In an assay for vitamin B₁₂ whereina serum sample is contacted with vitamin B₁₂ tracer and vitamin B₁₂binder, the improvement comprising:initially releasing vitamin B₁₂ fromendogenous binders therefor by contacting the serum with a vitamin B₁₂releasing agent comprising a water miscible organic liquid solvent whichdoes not destroy vitamin B₁₂, a reducing agent which does not destroyvitamin B₁₂, and cyanide ions, said releasing agent being employed in anamount effective to release vitamin B₁₂ from endogenous binders thereforto thereby eliminate the necessity for heating the serum to heatreleasing temperatures.
 9. The assay of claim 8 wherein the ratio ofreducing agent to organic liquid solvent is from 0.00001:1 to 0.1:1 andthe ratio of cyanide ion to organic liquid solvent is from 0.00001:1 to0.1:1.
 10. The assay of claim 9 wherein the organic liquid solvent isselected from the group consisting of ketones, alcohols, ethers,dimethylformamide and dimethylsulfoxide.
 11. The assay of claim 10wherein the cyanide ion to organic liquid ratio is from 0.00025:1 to0.00075:1 and the reducing agent to organic liquid ratio is from0.00025:1 to 0.00075:1.
 12. The assay of claim 11 wherein the releasingagent is employed as an aqueous solution wherein the ratio of organicliquid to water is from 4:1 to 6:1.
 13. The assay of claim 11 whereinthe releasing agent is employed in an amount to provide a ratio oforganic liquid to serum of at least 0.5:1 and no greater than 10:1. 14.The assay of claim 13 wherein the organic liquid is acetone and thereducing agent is dithiothreitol.
 15. A kit for the assay of vitaminB₁₂, comprising:vitamin B₁₂ tracer, vitamin B₁₂ binder and a vitamin B₁₂releasing agent comprising a water miscible organic liquid solvent whichdoes not destroy vitamin B₁₂, a reducing agent which does not destroyvitamin B₁₂, and cyanide ions, said releasing agent being employed in anamount effective to release vitamin B₁₂ from endogenous binders thereforwithout the necessity for heating a serum sample to heat releasingtemperatures.
 16. The kit of claim 15 wherein the ratio of reducingagent to organic liquid solvent is from 0.00001:1 to 0.1:1 and the ratioof cyanide ion to organic liquid solvent is from 0.00001:1 to 0.1:1. 17.The kit of claim 16 wherein the organic liquid solvent is selected fromthe group consisting of ketones, alcohols, ethers, dimethylformamide anddimethylsulfoxide.
 18. The kit of claim 17 wherein the releasing agentis employed as an aqueous solution wherein the ratio of organic liquidto water is from 4:1 to 6:1.
 19. The kit of claim 18 wherein the organicliquid is acetone and the reducing agent is dithiothreitol.
 20. The kitof claim 16 wherein the vitamin B₁₂ tracer is radiolabeled vitamin B₁₂.21. The kit of claim 20 wherein the vitamin B₁₂ tracer is vitamin B₁₂[⁵⁷ Co].
 22. The kit of claim 21 wherein the vitamin B₁₂ binder is hogintrinsic factor.
 23. The kit of claim 22 wherein the organic liquid isacetone and the reducing agent is dithiothreitol.
 24. The kit of claim23 wherein the releasing agent is employed as an aqueous solutionwherein the ratio of acetone to water is from 4:1 to 6:1.
 25. The kit ofclaim 24 wherein the cyanide ion to acetone ratio is from 0.00025:1 to0.00075:1 and the dithiothreitol to acetone ratio is from 0.00025:1 to0.00075:1.
 26. The process of claim 1 wherein the contacting is effectedat a temperature of less than 100° F.
 27. The process of claim 26wherein the contacting is effected at a temperature of from roomtemperature to 37° C.
 28. The process of claim 1 wherein the reducingagent is selected from the group consisting of dithiothreitol,dithioerythritol, monothioglycol, thiodiglycol, thioglycollic acid,cysteine, homocysteine, glutathione, mercaptoethanol, sulfhydrylreducing agents, sodium sulfite, sodium dithionate, sodium sulfide andsodium metabisulfite.
 29. The assay of claim 8 wherein the contacting iseffected at a temperature of less than 100° F.
 30. The assay of claim 29wherein the contacting is effected at a temperature of from roomtemperature to 37° C.
 31. The assay of claim 8 wherein the reducingagent is selected from the group consisting of dithiothreitol,dithioerythritol, monothioglycol, thiodiglycol, thioglycollic acid,cysteine, homocysteine, glutathione, mercaptoethanol, sulfhydrylreducing agents, sodium sulfite, sodium dithionate, sodium sulfide andsodium metabisulfite.
 32. The kit of claim 17 wherein the reducing agentis selected from the group consisting of dithiothreitol,dithioerythritol, monothioglycol, thiodiglycol, thioglycollic acid,cysteine, homocysteine, glutathione, mercaptoethanol, sulfhydrylreducing agent, sodium sulfite, sodium dithionate, sodium sulfide andsodium metabisulfite.